Abstract The nucleus is compartmentalized into domains termed nuclear bodies, which serve to properly coordinate various gene expression pathways. These pathways are often targeted by human pathogens or disrupted in other diseases. However, there is limited knowledge regarding the structure and function of nuclear bodies. Strikingly, the influenza virus subverts nuclear speckles, an intranuclear compartment involved in RNA processing, to splice the viral M1 mRNA to generate M2 mRNA. The unspliced M1 mRNA segment generates the M1 matrix protein whereas removal of an internal intron in the M1 transcript leads to the M2 form of the mRNA, which encodes an ion channel. Both M1 and M2 proteins are essential for viral trafficking and budding, thus the nuclear speckle-associated splicing of M1 to M2 mRNA is a critical aspect of the viral life cycle. It has recently been shown that the cellular proteins NS1-BP and hnRNP K form a complex to mediate M1 mRNA splicing and specifically yield the M2 mRNA. Importantly, depletion of either NS1-BP or hnRNP K perturbs the association of the M1/M2 mRNAs with nuclear speckles, while disruption of speckle integrity impedes M1 to M2 splicing. Moreover, the influenza virulence protein NS1, which binds to NS1-BP, also promotes M1 speckle localization and splicing. By contrast, inhibition of speckle function by depletion of the core speckle protein SON, inhibits M2 production and viral replication. Thus, the splicing of M1 mRNA to M2 mRNA is directly associated with nuclear speckles. Since nuclear speckles are not usually sites for splicing but are storage sites of splicing factors, the M1 to M2 splicing at nuclear speckles represents a new intranuclear trafficking pathway that may represent a novel opportunity for antiviral therapy. This proposal leverages a multi-pronged approach involving cell biology, RNA biochemistry, virology and structural biology to determine the mechanisms through which NS1-BP, hnRNP K and NS1, and perhaps additional proteins, regulate nuclear trafficking and promote pre-mRNA splicing at speckles. High-resolution and live cell imaging will be used to determine the protein factors and RNA sequences that mediate nuclear transport and speckle localization of the influenza M1/M2 RNAs. Parallel studies will be done to determine the impact of the same RNA sequences on the binding of NS1, NS1-BP and hnRNP K to the M1 RNA, and the impact of these sequences and associated proteins on M1 to M2 splicing. Genomic approaches will also be used to determine if a subset of host genes are spliced by a similar pathway as M1 in either normal or influenza- infected cells. Finally, the interaction of NS1-BP with NS1 and hnRNP K will be studied in atomic-level detail by crystallization of individual domains and protein complexes. Together these studies will uncover novel mechanisms of, and connections between, alternative splicing and nuclear transport and how these processes are subverted by the influenza virus. As such, the studies described here will reveal host vulnerabilities targeted by influenza virus that can potentially be used to devise new therapeutic options.